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               E.coli news vol. 14             2006.12.8
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This is a digest of daily PubMed searching of E.coli new finding.

***this year finding***

 >>>YeeI/mtfA, PTS system candidate, Mlc binding protein<<<
J Bacteriol. 2006 Aug;188(15):5439-49.
YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12.
Becker AK, Zeppenfeld T, Staab A, Seitz S, Boos W, Morita T, Aiba H, Mahr K, Titgemeyer F, Jahreis K.
Department of Biology and Chemistry, University of Osnabruck, D-49069 Osnabruck, Germany.

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced i
nduction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.

PMID: 16855233


 >>>frmB, yeiG, S-formylglutathione hydrolase<<<
J Biol Chem. 2006 Mar 27; [Epub ahead of print]
Molecular basis of formaldehyde detoxification: Characterization of two s-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG.
Gonzalez CF, Proudfoot M, Brown G, Korniyenko Y, Mori H, Savchenko AV, Yakunin AF.
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6.

The E. coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2 to C6) and a-naphthyl acetate. However, both proteins had highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione h
ydrolase YJG8 (PDB code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical a/b-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.

PMID: 16567800


 >>>AcrA, multidrug efflux system protein<<<
Structure. 2006 Mar;14(3):577-87.
Conformational flexibility in the multidrug efflux system protein AcrA.
Mikolosko J, Bobyk K, Zgurskaya HI, Ghosh P.
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92037, USA.

Intrinsic resistance to multiple drugs in many gram-negative bacterial pathogens is conferred by resistance nodulation cell division efflux pumps, which are composed of three essential components as typified by the extensively characterized Escherichia coli AcrA-AcrB-TolC system. The inner membrane drug:proton antiporter AcrB and the outer membrane channel TolC export chemically diverse compounds out of the bacterial cell, and require the activity of the third component, the periplasmic protein AcrA. The crystal structures of AcrB and TolC have previously been determined, and we complete the molecular picture of the efflux system by presenting the structure of a stable fragment of AcrA. The AcrA fragment resembles the elongated sickle shape of its homolog Pseudomonas aeruginosa MexA, being composed of three domains: beta-barrel, lipoyl, and alpha-helical hairpin. Notably, unsuspected conformational flexibility in the alpha-helical hairpin domain of AcrA is observed
, which has potential mechanistic significance in coupling between AcrA conformations and TolC channel opening.

PMID: 16531241

***additional finding***


 >>>yoeB, RNase<<<
Mol Cell. 2005 Aug 19;19(4):497-509.
Conformational change in the catalytic site of the ribonuclease YoeB toxin by YefM antitoxin.Kamada K, Hanaoka F.
Cellular Physiology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. kamadak@riken.jp

The eubacterial chromosome encodes various addiction modules that control global levels of translation through RNA degradation. Crystal structures of the Escherichia coli YefM2 (antitoxin)-YoeB (toxin) complex and the free YoeB toxin have been determined. The structure of the heterotrimeric complex reveals an asymmetric disorder-to-order recognition strategy, in which one C terminus of the YefM homodimer exclusively interacts with an atypical microbial ribonuclease (RNase) fold of YoeB. Comparison with the YefM-free YoeB structure indicates a conformational rearrangement of the RNase catalytic site of YoeB, induced by interaction with YefM. Complementary biochemical experiments demonstrate that the YoeB toxin has an in vitro RNase activity that preferentially cleaves at the 3' end of purine ribonucleotides.

PMID: 16109374


 >>>AcrB, multidrug efflux pump<<<
J Bacteriol. 2005 Oct;187(19):6804-15.
A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study.
Yu EW, Aires JR, McDermott G, Nikaido H.
Department of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, CA 94720-3202, USA.

The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-beta-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specifici
ty. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.

PMID: 16166543


 >>>yrdA, spermine induced protein<<<
BMC Microbiol. 2005 Oct 13;5:59.
Polyamine stress at high pH in Escherichia coli K-12.
Yohannes E, Thurber AE, Wilks JC, Tate DP, Slonczewski JL.
Department of Biology, Kenyon College, Gambier, OH 43022, USA. yohannese@kenyon.edu

BACKGROUND: Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress. RESULTS: The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range). At pH 2, 10 mM spermine increased survival by 2-fold, and putrescine increased survival by 30%. At pH 9.8, however, E. coli survival was decreased 100-fold by 10 mM spermine, putrescine, cadaverine, or spermidine. At pH 8.5, spermine decreased the growth rate substantially, whereas little effect was seen at pH 5.5. Sperm
idine required ten-fold higher concentrations to impair growth. On proteomic 2-D gels, spermine and spermidine caused differential expression of 31 different proteins. During log-phase growth at pH 7.0, 1 mM spermine induced eight proteins, including PykF, GlpK, SerS, DeaD, OmpC and OmpF. Proteins repressed included acetate-inducible enzymes (YfiD, Pta, Lpd) as well as RapA (HepA), and FabB. At pH 8.5, spermine induced additional proteins: TnaA, OmpA, YrdA and NanA (YhcJ) and also repressed 17 proteins. Four of the proteins that spermine induced (GlpK, OmpA, OmpF, TnaA) and five that were repressed (Lpd, Pta, SucB, TpiA, YfiD) show similar induction or repression, respectively, in base compared to acid. Most of these base stress proteins were also regulated by spermidine, but only at ten-fold higher concentration (10 mM) at high pH (pH 8.5). CONCLUSION: Polyamines increase survival in extreme acid, but decrease E. coli survival in extreme base. Growth inhibition by
 spermine and spermidine requires neutral or higher pH. At or above pH 7, spermine and spermidine regulate specific proteins, many of which are known to be regulated by base stress. High pH amplifies polyamine stress; and naturally occurring polyamines may play an important role in base stress.

PMID: 16223443


 >>>yadF/can, carbonic anhydrase<<<
J Bacteriol. 2003 Nov;185(21):6415-24.
Why is carbonic anhydrase essential to Escherichia coli?
Merlin C, Masters M, McAteer S, Coulson A.
University of Edinburgh, Institute of Cell and Molecular Biology, Edinburgh EH9 3JR, Scotland.

The can (previously yadF) gene of Escherichia coli encodes a beta-class carbonic anhydrase (CA), an enzyme which interconverts CO(2) and bicarbonate.Various essential metabolic processes require either CO(2) or bicarbonate and, although carbon dioxide and bicarbonate spontaneously equilibrate in solution, the low concentration of CO(2) in air and its rapid diffusion from the cell mean that insufficient bicarbonate is spontaneously made in vivo to meet metabolic and biosynthetic needs. We calculate that demand for bicarbonate is 10(3)- to 10(4)-fold greater than would be provided by uncatalyzed intracellular hydration and that enzymatic conversion of CO(2) to bicarbonate is therefore necessary for growth. We find that can expression is ordinarily required for growth in air. It is dispensable if the atmospheric partial pressure of CO(2) is high or during anaerobic growth in a closed vessel at low pH, where copious CO(2) is generated endogenously. CynT, the single E.
coli Can paralog, can, when induced with azide, replace Can; also, the gamma-CA from Methanosarcina thermophila can at least partially replace it. Expression studies showed that can transcription does not appear to respond to carbon dioxide concentration or to be autoregulated. However, can expression is influenced by growth rate and the growth cycle; it is expressed best in slow-growing cultures and at higher culture densities. Expression can vary over a 10-fold range during the growth cycle and is also elevated during starvation or heat stress.

PMID: 14563877

Biosci Biotechnol Biochem. 2003 Apr;67(4):919-22.
Indispensability of the Escherichia coli carbonic anhydrases YadF and CynT in cell proliferation at a low CO2 partial pressure.
Hashimoto M, Kato J.
Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan.

We found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3-. This suggests that these carbonic anhydrases are essential because they maintain HCO3- levels at ambient CO2 concentrations.

PMID: 12784642

 >>>MazF, sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA<<<
Mol Cell. 2003 Oct;12(4):913-23.  Links
MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli.Zhang Y, Zhang J, Hoeflich KP, Ikura M, Qing G, Inouye M.
Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.

Escherichia coli contains operons called "addiction modules," encoding toxin and antitoxin, which are responsible for growth arrest and cell death. Here, we demonstrate that MazF toxin encoded by "mazEF addiction module" is a sequence-specific (ACA) endoribonuclease functional only for single-stranded RNA. MazF works as a ribonuclease independent of ribosomes, and is, therefore, functionally distinct from RelE, another E. coli toxin, which assists mRNA cleavage at the A site on ribosomes. Upon induction, MazF cleaves whole cellular mRNAs to efficiently block protein synthesis. Purified MazF inhibited protein synthesis in both prokaryotic and eukaryotic cell-free systems. This inhibition was released by MazE, the labile antitoxin against MazF. Thus, MazF functions as a toxic endoribonuclease to interfere with the function of cellular mRNAs by cleaving them at specific sequences leading to rapid cell growth arrest and cell death. The role of such endoribonucleases ma
y have broad implication in cell physiology under various growth conditions.

PMID: 14580342